1. Field of the Invention
This invention relates generally to methods for the synthesis of phosphonate esters. More particularly, the invention provides methods for producing monoesters and diesters of phosphonic acids from condensations of alcohols with alkylphosphonates, such as methyl alphaaminoalkylphosphonates. The methods can be carried out in solution phase, but in an important embodiment, the method is carried out on a solid support, such as beads or a glass slide, and used to create large arrays of diverse compounds, such as peptidylphosphonates and polyphosphonates, which can then be conveniently screened for the presence of compounds with desired pharmacological or other properties. The invention therefore relates to the fields of chemistry, enzymology, pharmacology, and medicinal chemistry.
2. Description of Related Art
Phosphonic acid monoesters have typically been synthesized by one of two methods. In the first, a monomethyl alkylphosphoryl chloride is treated with an alcohol. Selective de-esterification of the methyl ester yields the desired product. The monomethyl alkylphosphoryl chloride is produced by the direct action of PCl.sub.5 on a phosphonate diester (see Balthazor et al., 1980, J. Org. Chem. 45: 530) or by base hydrolysis of the phosphonate diester followed by reaction with thionyl chloride (see Bartlett et al., 1990, Org. Chem. 55: 6268).
Alternatively, the direct monoesterification of a phosphonic acid can be accomplished with the appropriate alcohol and condensing reagents (see Gilmore et al., 1974, J. Pharm. Sci. 63: 965), such as 1,3-dicyclohexylcarbodiimide (DCC) or trichloroacetonitrile (see, e.g., Wasielewski et al., 1976, J. Rocz. Chem. 50: 1613). A large excess of both the condensing agent and alcohol is required, and yields vary depending on the components being coupled. In addition, forcing conditions are required, such as heating to reflux temperature in a solution of THF with triethylamine and DCC, and to 50.degree. C.-80.degree. C. in pyridine with trichloroacetonitrile, conditions that can lead to decomposition of starting material. In addition, product yields are extremely sensitive to steric encumbrance from the reacting components, and in some cases, no product formation is observed.
These phosphonate synthesis procedures are not suitable for solid phase chemical synthesis methods, which presents a significant problem for the medicinal chemist. Peptidylphosphonates are peptide analogues that have been used extensively as inhibitors of metalloproteases and other enzymes and as haptens in the generation of catalytic antibodies. The most potent non-covalent enzyme inhibitor known is a phosphonyltripeptide inhibitor of carboxypeptidase A, which binds with 11 femptomolar (fM) K.sub.d. See Kaplan et al., 1991, Biochem. 30: 8165-8170. To synthesize these derivatives on a solid support, one needs a general method for introducing the phosphonic acid monoester functionality.
The Mitsunobu reaction is a mild and effective method utilizing the redox chemistry of triphenylphosphine and a dialkylazodicarboxylate to condense an acidic reagent with primary and secondary alcohols (see Mitsunobu, 1981, Synthesis 1-28). The Mitsunobu reference does not describe the reaction of phosphate esters with secondary or tertiary alcohols or the reaction of phosphonates with alcohols. The Mitsunobu reaction has been used with carboxylic acids, phenols, and phosphates as the acidic component, but only one example using a phosphonic acid has been described.
In this example (see Norbeck et al., 1987, J. Org. Chem. 52: 2174), a monomethyl phosphonate and a primary alcohol were reacted under modified Mitsunobu conditions (triphenylphosphine and diisopropylazodicarboxylate) to produce a phosphonate diester. Demethylation using triethylamine and thiophenol produced the phosphonate monoester. Yields, however, were low for the condensation of a phosphate monoester with even a primary alcohol. Only moderate yields were obtained with elevated temperatures or with hexamethylphosphorous triamide (HMPT) as the solvent.
Thus, there remains a need for a general method for synthesizing phosphonate esters. Additional examples of the utility of peptidylphosphonate esters are numerous; these compounds are recognized as effective transition-state analogue inhibitors for a variety of enzymes, including a number of other proteases and esterases (see, e.g., Morgan et al., 1991, J. Am. Chem. Soc. 113: 297 and Bartlett et al., 1990, J. Org. Chem. 55: 6268). Peptidylphosphonate esters have been used as non-hydrolyzable analogues of phosphates to inhibit dinucleoside triphosphate hydrolase (see, e.g., Blackburn et al., 1987, Nucl. Acids Res. 15: 6991), phosphatidyltransferase (see, e.g., Vargas et al., 1984, Biochim. Biophys. Acta 796: 123), and squalene synthetase (see, e.g., Biller et al., 1988, J. Meal. Chem. 31: 1869). In addition, phosphonate esters have been used as haptens for the production of catalytic antibodies possessing esterase activity (see, e.g., Jacobs et al., 1987, J. Am. Chem. Soc. 109: 2174; Tramontanio et al., 1986, Science 234: 1566; and Pollack et al., 1986, Science 234: 1570; see also, U.S. patent application Ser. No. 858,298, filed Mar. 26, 1992).
The solution phase synthesis of these derivatives, such as a relatively short tetramer, can take weeks to months to complete by prior art methods. If one attempts to synthesize and screen a large number of such compounds, then the synthesis time required can make the project impossible to complete. Recently, an innovative method for the synthesis of large numbers of polymeric compounds on the surface of a solid support has been developed. This method, called VLSIPS.TM. technology, is described in U.S. patent application Ser. No. 805,727, filed Dec. 6, 1991, which is a continuation-in-part of Ser. No. 624,120, filed Dec. 6, 1990, which is a continuation-in-part of U.S. Pat. No. 5,143,854, which is a continuation-in-part of Ser. No. 362,901, filed Jun. 7, 1989, and now abandoned. There remains a need for chemical synthesis methods for use in the VLSIPS.TM. method to generate and screen large numbers of peptidylphosphonates and other compounds containing phosphonate ester building blocks.
In similar fashion, there remains a need for a mild coupling reaction for the preparation of phosphonate esters that is compatible with a variety of functional groups and consistently produces high yields of the desired compounds and that can be applied to solution as well as solid phase chemistry. The present invention meets these needs.